A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

Handle URI:
http://hdl.handle.net/10754/325317
Title:
A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling
Authors:
Aouida, Mustapha ( 0000-0001-7945-5320 ) ; Kim, Kangchang; Shaikh, Abdul Rajjak; Pardo, Jose M.; Eppinger, Jorg ( 0000-0001-7886-7059 ) ; Yun, Dae-Jin; Bressan, Ray A.; Narasimhan, Meena L.
Abstract:
Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology. 2013 Aouida et al.
KAUST Department:
Plant Stress Genomics Research Lab; KAUST Catalysis Center (KCC); Physical Sciences and Engineering (PSE) Division; Center for Desert Agriculture
Citation:
Aouida M, Kim K, Shaikh AR, Pardo JM, Eppinger J, et al. (2013) A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling. PLoS ONE 8: e65454. doi:10.1371/journal.pone.0065454.
Publisher:
Public Library of Science (PLoS)
Journal:
PLoS ONE
Issue Date:
7-Jun-2013
DOI:
10.1371/journal.pone.0065454
PubMed ID:
23762377
PubMed Central ID:
PMC3676391
Type:
Article
ISSN:
19326203
Appears in Collections:
Articles; Physical Sciences and Engineering (PSE) Division; Center for Desert Agriculture; KAUST Catalysis Center (KCC)

Full metadata record

DC FieldValue Language
dc.contributor.authorAouida, Mustaphaen
dc.contributor.authorKim, Kangchangen
dc.contributor.authorShaikh, Abdul Rajjaken
dc.contributor.authorPardo, Jose M.en
dc.contributor.authorEppinger, Jorgen
dc.contributor.authorYun, Dae-Jinen
dc.contributor.authorBressan, Ray A.en
dc.contributor.authorNarasimhan, Meena L.en
dc.date.accessioned2014-08-27T09:46:40Zen
dc.date.available2014-08-27T09:46:40Zen
dc.date.issued2013-06-07en
dc.identifier.citationAouida M, Kim K, Shaikh AR, Pardo JM, Eppinger J, et al. (2013) A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling. PLoS ONE 8: e65454. doi:10.1371/journal.pone.0065454.en
dc.identifier.issn19326203en
dc.identifier.pmid23762377en
dc.identifier.doi10.1371/journal.pone.0065454en
dc.identifier.urihttp://hdl.handle.net/10754/325317en
dc.description.abstractAdiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology. 2013 Aouida et al.en
dc.language.isoenen
dc.publisherPublic Library of Science (PLoS)en
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en
dc.rightsArchived with thanks to PLoS ONEen
dc.subjectadiponectinen
dc.subjectadiponectin receptor 1en
dc.subjectcyclic AMP dependent protein kinaseen
dc.subjecthydroxymethylglutaryl coenzyme A reductase kinaseen
dc.subjectbinding affinityen
dc.subjectbioassayen
dc.subjectcell suspensionen
dc.subjectcomplex formationen
dc.subjectcontrolled studyen
dc.subjectenzyme activationen
dc.subjectenzyme phosphorylationen
dc.subjectfungal strainen
dc.subjectmolecular interactionen
dc.subjectprotein functionen
dc.subjectquantitative analysisen
dc.subjectreceptor bindingen
dc.subjectSaccharomyces cerevisiaeen
dc.subjectsignal transductionen
dc.subjectAdiponectinen
dc.subjectAntigens, Planten
dc.subjectBiological Assayen
dc.subjectCell Membraneen
dc.subjectGene Expression Regulation, Fungalen
dc.subjectGenes, Reporteren
dc.subjectLigandsen
dc.subjectLuciferases, Fireflyen
dc.subjectMolecular Docking Simulationen
dc.subjectPeptidesen
dc.subjectPhosphorylationen
dc.subjectPlant Proteinsen
dc.subjectPromoter Regions, Geneticen
dc.subjectProtein-Serine-Threonine Kinasesen
dc.subjectReceptors, Adiponectinen
dc.subjectSaccharomyces cerevisiaeen
dc.subjectSignal Transductionen
dc.subjectMammaliaen
dc.subjectNicotiana tabacumen
dc.subjectSaccharomyces cerevisiaeen
dc.titleA Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signalingen
dc.typeArticleen
dc.contributor.departmentPlant Stress Genomics Research Laben
dc.contributor.departmentKAUST Catalysis Center (KCC)en
dc.contributor.departmentPhysical Sciences and Engineering (PSE) Divisionen
dc.contributor.departmentCenter for Desert Agricultureen
dc.identifier.journalPLoS ONEen
dc.identifier.pmcidPMC3676391en
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionDivision of Applied Life Science (Brain Korea 21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Koreaen
dc.contributor.institutionInstituto de Recursos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientficas, Sevilla, Spainen
dc.contributor.institutionDepartment of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, United Statesen
dc.contributor.institutionBio-Write LLC, West Lafayette, IN, United Statesen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
kaust.authorAouida, Mustaphaen
kaust.authorShaikh, Abdul Rajjaken
kaust.authorEppinger, Jorgen
kaust.authorBressan, Ray Anthonyen
kaust.authorNarasimhan, Meena L.en

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